Template Generation System™ I Kit, TGS™ I Kit

Transposon-mediated insertion of primer binding sites into target DNA for sequencing or PCR* amplification

Description

The Template Generation System™ I Kit (TGS™ I Kit) is a powerful tool for generating templates for sequencing and PCR amplification based on the in vitro transposition reaction of the bacteriophage Mu. The replicative transposition of the bacteriophage Mu during the lytic phase is one of the most effective DNA mobilization events that has been studied in detail. The Template Generation System I Kit provides the tools for inserting the artificial Mu transposon, designated as the Entranceposon, into foreign DNA at random locations in a simple in vitro reaction catalyzed by a single enzyme, MuA Transposase.

Components

The kit contains sufficient materials for 20 reactions:

Kit components
MuA Transposase	20 µl	(0.22 µg/µl in MuA storage buffer)
Entranceposon (CamR)	20 µl	(20 ng/µl in TE buffer)
Entranceposon (KanR)	20 µl	(20 ng/µl in TE buffer)
5x Reaction Buffer	100 µl
Control Target DNA	10 µl	(370 ng/µl in TE buffer)
MuEnd Primer	800 µl	(25 µM in dd water)
pUC Fwd Primer	400 µl	(25 µM in dd water)
pUC Rev Primer	400 µl	(25 µM in dd water)
Seq A Primer	250 µl	(10 µM in dd water)
Seq B Primer	250 µl	(10 µM in dd water)

Storage stability

Store the components at –20 °C. Stable for 1 year from the date of packaging when stored and handled properly.

System protocol

Day 1.

a. In vitro transposition reaction and transformation
Perform the transposition reaction; MuA Transposase catalyzes the in vitro transposition reaction in which the Entranceposons are inserted into the target DNA clones at random sites (70 minutes). Transform the DNA into competent E.coli cells, plate and cultivate the cells overnight.

Day 2.

On the day 2 there are three different options to proceed: a, b, c

a. Colony-PCR mapping and DNA sequencing from PCR product
Map the Entranceposon insertions in individual clones using colony-PCR and use the resulting PCR products directly as sequencing templates.

b. Colony-PCR mapping and plasmid preparation
Map the Entranceposon insertions in individual clones using colony-PCR and start overnight liquid cultures from the clones of your choice.

c. No mapping, plasmid preparation from randomly selected clones.
Start overnight liquid cultures from randomly selected clones (especially suitable for target DNA clones that are too large for colony-PCR mapping, e.g. BACs).

Day 3.

a. Analyze the DNA sequencing data
DNA sequencing using the products from the colony-PCR reactions as template is the fastest way to complete the sequence
b, c. Plasmid preparation and DNA sequencing

Ordering information

Template Generation System™ I Kit (TGS™ I Kit)
F-700	Template Generation System™ I Kit	For 20 reactions
F-750	MuA Transposase with 5x Reaction Buffer	20 µl (0.22 µg/µl in MuA Storage Buffer)
F-751	Entranceposon (CamR)	20 µl (20 ng/µl in TE buffer)
F-759	Entranceposon (KanR)	20 µl (20 ng/µl in TE buffer)

Instruction Manual for Template Generation System™ I Kit (TGS™ I Kit) Technical Appendix – Transposon Products FAQ – Transposon Products Transposon Products References

Template Generation System™ II Kit Transposon products available separately